Nested fluorescence reverse transcription-polymerase chain reaction (RT-PCR) detection method for avian influenza virus (AIV) H5 subtype and detection kit

Abstract

The invention discloses a nested fluorescence reverse transcription-polymerase chain reaction (RT-PCR) detection method for avian influenza virus (AIV) H5 subtype with high efficiency and high sensibility and a detection kit. In the method, two sets of polymerase chain reaction (PCR) primers (a pair of outer primers and a pair of inner primers) are utilized to carry out PCR amplification twice, that is, a product obtained after the outer primers are subjected to PCR amplification is taken as a template which is used for carrying out the PCR amplification on the inner primers; a binding site of the inner primers and the template DNA is located at the inner side of a DNA fragment which is obtained by amplifying the outer primers; the nested PCR is very effective in reducing or eliminating nonspecific amplification and improving sensitivity. Compared with common detection methods, the method disclosed by the invention has good specificity, and can exactly detect the subtype AIV of H5N1 and H5N2 and detect H1N1, H3N2, H6, H9NDVLasata and EDSV to be negative; is very beneficial to amplifying an AVI micro-template in a fish farming water body, and can completely meet the requirements onsensibility and specificity of AVI detection in the cultivation water of fishponds.

Description

technical field [0001] The invention relates to a nested fluorescent RT-PCR detection method and a detection kit for H5 subtype of avian influenza virus. Background technique [0002] The isolation and identification of avian influenza virus needs to collect throat swabs or cloacal swabs from birds, or collect internal organs from sick and dead birds, use chicken embryos for virus isolation and culture, and then identify hemagglutinin (HA) and neuraminic acid Enzyme (NA), and finally the determination of pathogenicity through artificial infection of chickens. This classic detection method of avian influenza virus is recommended by OIE as an international reference method due to its high sensitivity. It is carried out in a third-level laboratory, so it cannot be used for rapid detection, and the existing avian influenza virus detection methods, such as conventional RT-PCR technology, colloidal gold immunoassay and ELISA, cannot be directly used in poultry and poultry due to t...

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Problems solved by technology

[0002] The isolation and identification of avian influenza virus needs to collect throat swabs or cloacal swabs from birds, or collect internal organs from sick and dead birds, and use chicken embryos for virus Isolate and cultivate, then identify hemagglutinin (HA) and neuraminidase (NA), and finally measure the pathogenicity through artificial infection of chickens. This classic detection method for avian influenza virus is recommended by OIE as the most sensitive method because of its high sensitivity. International reference method, but due to the long diagnostic cycle and the need to carry out in a biosafety level 3 laboratory, it cannot be used for rapid detection, while the existing detection methods for avian influenza virus, such as conventional RT-PCR technology, colloidal gold immunoassay and ELISA, etc., cannot be directly used for the detection of AIV in poultry and poultry products due to their low sensitivity

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